U.S. patent application number 14/213243 was filed with the patent office on 2014-09-18 for fabrication of nanopores in atomically-thin membranes by ultra-short electrical pulsing.
This patent application is currently assigned to President and Fellows of Harvard College. The applicant listed for this patent is President and Fellows of Harvard College. Invention is credited to Jene A. Golovchenko, Aaron T. Kuan.
Application Number | 20140262820 14/213243 |
Document ID | / |
Family ID | 51522627 |
Filed Date | 2014-09-18 |
United States Patent
Application |
20140262820 |
Kind Code |
A1 |
Kuan; Aaron T. ; et
al. |
September 18, 2014 |
Fabrication of Nanopores In Atomically-Thin Membranes By
Ultra-Short Electrical Pulsing
Abstract
In a method for forming nanopores, two opposing surfaces of a
membrane are exposed to an electrically conducting liquid
environment. A nanopore nucleation voltage pulse, having a first
nucleation pulse amplitude and duration, is applied between the two
membrane surfaces, through the liquid environment. After applying
the nanopore nucleation voltage pulse, the electrical conductance
of the membrane is measured and compared to a first prespecified
electrical conductance. Then at least one additional nanopore
nucleation voltage pulse is applied between the two membrane
surfaces, through the liquid environment, if the measured
electrical conductance is no greater than the first prespecified
electrical conductance. At least one nanopore diameter tuning
voltage pulse, having a tuning pulse voltage amplitude and
duration, is applied between the two membrane surfaces, through the
liquid environment, if the measured electrical conductance is
greater than the first prespecified electrical conductance and no
greater than a second prespecified electrical conductance.
Inventors: |
Kuan; Aaron T.; (Cambridge,
MA) ; Golovchenko; Jene A.; (Lexington, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
President and Fellows of Harvard College |
Cambridge |
MA |
US |
|
|
Assignee: |
President and Fellows of Harvard
College
Cambridge
MA
|
Family ID: |
51522627 |
Appl. No.: |
14/213243 |
Filed: |
March 14, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61790089 |
Mar 15, 2013 |
|
|
|
Current U.S.
Class: |
205/665 |
Current CPC
Class: |
B01D 67/0034 20130101;
G01N 33/48721 20130101; B01D 2323/35 20130101; B01D 2325/02
20130101; C25F 3/14 20130101; C25F 3/00 20130101; B01D 69/02
20130101; B01D 67/0065 20130101; B05D 1/34 20130101; B01D 67/0062
20130101 |
Class at
Publication: |
205/665 |
International
Class: |
C25F 3/14 20060101
C25F003/14 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with Government support under
Contract No. 5R01HG003703 awarded by the National Institutes of
Health. The Government has certain rights in the invention.
Claims
1. A method for forming a nanopore in a membrane comprising:
exposing two opposing surfaces of a membrane to an electrically
conducting liquid environment; applying a nanopore nucleation
voltage pulse, having a first nucleation pulse amplitude, between
the two membrane surfaces, through the liquid environment, the
nanopore nucleation voltage pulse having a pulse duration; after
applying the nanopore nucleation voltage pulse, measuring the
electrical conductance of the membrane and comparing the measured
electrical conductance to a first prespecified electrical
conductance; applying at least one additional nanopore nucleation
voltage pulse between the two membrane surfaces, through the liquid
environment, each additional nanopore nucleation voltage pulse
being applied if the measured electrical conductance is no greater
than the first prespecified electrical conductance; and applying at
least one nanopore diameter tuning voltage pulse, having a tuning
pulse voltage amplitude, between the two membrane surfaces, through
the liquid environment, each nanopore diameter tuning voltage pulse
being applied if the measured electrical conductance is greater
than the first prespecified electrical conductance and no greater
than a second prespecified electrical conductance, each nanopore
diameter tuning voltage pulse having a pulse duration.
2. The method of claim 1 wherein applying at least one nanopore
diameter tuning voltage pulses comprises: applying a first nanopore
diameter tuning voltage pulse, having a first tuning pulse voltage
amplitude, between the two membrane surfaces, through the liquid
environment, the nanopore diameter tuning voltage pulse having a
pulse duration; after applying the first nanopore diameter tuning
voltage pulse, measuring the electrical conductance of the membrane
and comparing the measured electrical conductance to a third
prespecified electrical conductance; applying at least one
additional nanopore diameter tuning voltage pulse, having a second
tuning pulse voltage amplitude that is greater than the first
tuning pulse voltage amplitude, between the two membrane surfaces,
through the liquid environment, each additional nanopore diameter
tuning voltage pulse being applied if the measured electrical
conductance is no greater than the third prespecified electrical
conductance; and applying at least one additional nanopore diameter
tuning voltage pulse, having the first tuning pulse voltage
amplitude, between the two membrane surfaces, through the liquid
environment, the additional nanopore diameter tuning voltage pulse
being applied if the measured electrical conductance is greater
than the third prespecified electrical conductance and no greater
than the second prespecified electrical conductance.
3. The method of claim 1 wherein the pulse duration of each
nanopore nucleation voltage pulse and the pulse duration of each
nanopore diameter tuning voltage pulse is no greater than
250.times.10.sup.-9 second.
4. The method of claim 2 wherein the pulse duration of at least one
nanopore diameter tuning voltage pulse is less than a pulse
duration of a previously applied nanopore diameter tuning voltage
pulse.
5. The method of claim 1 wherein all nanopore nucleation voltage
pulses have an equal pulse duration.
6. The method of claim 1 wherein all nanopore diameter tuning
voltage pulses have an equal pulse duration.
7. The method of claim 1 wherein all nanopore nucleation voltage
pulses and all nanopore diameter tuning voltage pulses have an
equal pulse duration.
8. The method of claim 1 further comprising measuring electrical
leakage conductance of the membrane before applying a first
nanopore nucleation voltage pulse between the two membrane
surfaces, the measured electrical leakage conductance being
designated as the first prespecified electrical conductance.
9. The method of claim 1 wherein measuring electrical leakage
conductance of the membrane comprises applying a measurement
voltage pulse, having a measuring pulse amplitude, between the two
membrane surfaces, through the liquid environment, the measuring
pulse amplitude being less than 300 mV.
10. The method of claim 1 wherein applying at least one additional
nanopore nucleation voltage pulse between the two membrane surfaces
comprises applying at least one additional nanopore nucleation
voltage pulse having a second nucleation pulse amplitude that is
greater than the first nucleation pulse amplitude, through the
liquid environment, if the measured electrical conductance is less
than the first prespecified electrical conductance.
11. The method of claim 10 wherein each additional nucleation pulse
amplitude is at least 1 V greater than the a previous nucleation
pulse amplitude.
12. The method of claim 1 wherein each additional nanopore
nucleation voltage pulse is applied and the electrical conductance
of the membrane measured until the measured electrical conductance
is greater than the first prespecified electrical conductance.
13. The method of claim 1 wherein the second prespecified
electrical conductance corresponds to a membrane electrical
conductance for a membrane including a nanopore of a prespecified
diameter.
14. The method of claim 2 wherein the third prespecified electrical
conductance is greater than the first prespecified electrical
conductance.
15. The method of claim 2 wherein the third prespecified electrical
conductance is the electrical conductance measured before
application of a first nanopore diameter tuning voltage pulse.
16. The method of claim 1 wherein the membrane comprises an
atomically-thin material of no more than 50 nm in thickness.
17. The method of claim 1 wherein the membrane comprises
graphene.
18. The method of claim 1 wherein the electrically conducting
liquid environment comprises an electrolytic solution.
19. The method of claim 1 further comprising first processing the
membrane to produce at least one localized membrane site having a
material condition that imposes nanopore nucleation at that
site.
20. The method of claim 1 further comprising including in the
electrically conducting liquid environment a chemical species to
modify at least one of the membrane and a nucleated nanopore.
21. The method of claim 1 further comprising: after applying at
least one nanopore diameter tuning voltage pulse, adding to the
liquid environment, on one side of the two opposing membrane
surfaces, a polymer molecule species; and applying a translocation
voltage through the liquid environment, between the two opposing
membrane surfaces, to cause the polymer molecule species to
translocate through a nanopore in the membrane.
22. The method of claim 21 wherein the polymer molecule species
comprises at least a portion of a DNA strand.
23. A method for forming a nanopore in a membrane comprising:
exposing two opposing surfaces of a membrane to an electrically
conducting liquid environment; applying at least one nanopore
nucleation voltage pulse, having a nucleation pulse amplitude and
pulse duration, between the two membrane surfaces, through the
liquid environment, to produce at the membrane a nanopore
nucleation site; and after applying the nanopore nucleation voltage
pulse, processing the membrane to produce a preselected nanopore
diameter at the nanopore nucleation site.
24. The method of claim 23 wherein the membrane comprises
graphene.
25. The method of claim 23 wherein the membrane has a thickness
less than 50 nm.
26. The method of claim 23 wherein the at least one nanopore
nucleation voltage pulse has a pulse duration that is no greater
than 250.times.10.sup.-9 seconds.
27. A method for forming a nanopore in a membrane comprising:
exposing two opposing surfaces of a membrane to an electrically
conducting liquid environment; applying at least two nanopore
nucleation voltage pulses, each having a nucleation pulse amplitude
and pulse duration, between the two membrane surfaces, through the
liquid environment; and applying at least two nanopore diameter
tuning voltage pulses, each having a tuning voltage pulse amplitude
and pulse duration, between the two membrane surfaces, through the
liquid environment.
28. The method of claim 27 wherein all nanopore nucleation voltage
pulses and all nanopore diameter tuning voltage pulses have equal
pulse durations.
29. The method of claim 27 wherein each nanopore diameter tuning
voltage pulse has a tuning voltage pulse amplitude that is greater
than the tuning voltage pulse amplitude of a previously applied
tuning voltage pulse.
30. The method of claim 27 wherein membrane comprises graphene.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 61/790,089, filed Mar. 15, 2013, the entirety of
which is hereby incorporated by reference.
BACKGROUND
[0003] This invention relates generally to techniques for producing
apertures in thin materials, and more particularly relates to the
production of nanopores.
[0004] Nano-scale apertures, herein termed nanopores, have
attracted much research interest because a nanopore that is
provided through the thickness of a nanometrically-thin membrane
offers the ability to achieve exceptional resolution for a wide
range of molecular sensing and analysis applications, such as DNA
sequencing applications, and enables very little flow resistance
when employed for, e.g., nano-filtration applications that are in
graphene or other very-thin-material membranes, defined here as
being of a thickness that is less than about 50 nm, and can be less
than 5 nanometers. However, the fabrication of nanopores in such
atomically-thin materials, has conventionally be conducted by,
e.g., drilling with a focused electron beam. Such processing is
tedious, expensive, and can be low-yield because each nanopore must
be individually fabricated, e.g., in a high-resolution electron
microscope, and because the nanopore size is difficult to tune. In
addition, hydrocarbon contamination can be introduced and can
accumulate in and around a nanopore during electron beam pore
drilling. Such contamination, as well as the inherent surface
condition of the material around a nanopore, can render the
nanopore hydrophobic, thereby prohibiting wetting of the nanopore.
As a result, the reliable, repeatable production of functional
nanopores remains a fundamental nanotechnology challenge.
SUMMARY
[0005] The many limitations of prior approaches for forming
nanopores in nano-scale materials are overcome with a method for
forming nanopores that employs a ultra-short electrical pulsing. In
one such method, two opposing surfaces of a membrane are exposed to
an electrically conducting liquid environment. A nanopore
nucleation voltage pulse, having a first nucleation pulse
amplitude, is applied between the two membrane surfaces, through
the liquid environment. The nanopore nucleation voltage pulse has a
pulse duration. After applying the nanopore nucleation voltage
pulse, the electrical conductance of the membrane is measured and
compared to a first prespecified electrical conductance. Then at
least one additional nanopore nucleation voltage pulse is applied
between the two membrane surfaces, through the liquid environment,
if the measured electrical conductance is no greater than the first
prespecified electrical conductance. At least one nanopore diameter
tuning voltage pulse, having a tuning pulse voltage amplitude, is
applied between the two membrane surfaces, through the liquid
environment, if the measured electrical conductance is greater than
the first prespecified electrical conductance and no greater than a
second prespecified electrical conductance. Each nanopore diameter
tuning voltage pulse has a pulse duration.
[0006] In a further method for forming a nanopore, two opposing
surfaces of a membrane are exposed to an electrically conducting
liquid environment, and at least one nanopore nucleation voltage
pulse, having a nucleation pulse amplitude and pulse duration, is
applied between the two membrane surfaces, through the liquid
environment, to produce at the membrane a nanopore nucleation site.
After applying the nanopore nucleation voltage pulse, the membrane
is processed to produce a preselected nanopore diameter at the
nanopore nucleation site.
[0007] In a further method for forming a nanopore, two opposing
surfaces of a membrane are exposed to an electrically conducting
liquid environment. At least two nanopore nucleation voltage
pulses, each having a nucleation pulse amplitude and pulse
duration, are applied between the two membrane surfaces, through
the liquid environment. At least two nanopore diameter tuning
voltage pulses, each having a tuning voltage pulse amplitude and
pulse duration, are then applied between the two membrane surfaces,
through the liquid environment.
[0008] These methods enable very efficient, effective,
reproducible, and precise nanopore nucleation and tuning, in an
environment that maintains the nanopore wetted and free from
contamination. The methods are superior for enabling a wide range
of nanopore applications in which translocation of a species
through a pristine and well-defined nanopore is required. Other
features and advantages will be apparent from the following
description and accompanying figures, and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIG. 1 is a schematic side view of an electrically
conducting liquid environment for conducing the nanopore
fabrication process;
[0010] FIGS. 2A and 2B are schematic cross-sectional and top-down
views, respectively, of a graphene layer arranged with a support
frame structure for nanopore fabrication in the graphene;
[0011] FIG. 3 is a schematic view of a dual chamber flow cell for
implementing the liquid environment of FIG. 1 for a nanopore
fabrication process;
[0012] FIG. 4A is plot of nucleation voltage pulse duration for a
sequence of nucleation voltage pulses applied in the liquid
environment of FIG. 1 for nucleating a nanopore in an
atomically-thin membrane;
[0013] FIG. 4B is plot of tuning voltage pulse duration for a
sequence of tuning voltage pulses applied in the liquid environment
of FIG. 1 for enlarging a nanopore that has been nucleated in an
atomically-thin membrane;
[0014] FIG. 5 is plot of nucleation voltage pulse duration and
tuning voltage pulse duration for a sequence of nucleation and
tuning voltage pulses that were experimentally applied to a
graphene membrane in the liquid environment of FIG. 1 for
nucleating and enlarging a nanopore in a graphene membrane;
[0015] FIG. 6 is a plot of nanopore diameter calculated from
electrical current for the nanopore nucleation and enlarging
process depicted in the plot of FIG. 5, shown as a function of
applied voltage pulse number.
[0016] FIG. 7 is a plot of measured electrical current as a
function of time during an experimental process of translocating
DNA through a nanopore fabricated by the nanopore nucleation and
enlarging process depicted in the plot of FIG. 5.
DETAILED DESCRIPTION
[0017] Referring to FIG. 1, in the nanopore fabrication method
provided herein, there is configured an electrically conducting
fluid environment 10, with a membrane 12 of a selected material in
which one or more nanopores are to be produced being supported in
an electrically conducting fluid 14 by a support structure such as
a support frame 16. The membrane 12 is a continuous material that
is atomically thin, herein defined as a material that includes
between about one and about ten atomic layers of material and that
is no more than about 50 nm in thickness. Graphene, few-layer
graphene, multi-layer graphene, thin graphite, boron nitride,
molybdenum disulfide, thin mica, and silicene are examples of
atomically-thin materials that can be employed herein. The nanopore
fabrication method is particularly well-suited for such
atomically-thin materials and in general for membranes having a
thickness that is no more than about 5 nm. The membrane 12 can
include two or more material layers, including layers of differing
material composition. For example the membrane can include a layer
of graphene disposed on a layer of boron nitride. No particular
material or layers of materials are required. But for any material
layer composition, the complete thickness of the membrane is less
than about ten atomic layers and/or about 50 nanometers, and
preferably less than about 5 nanometers in thickness.
[0018] The membrane is mechanically self-supported across the
interior extent of the atomically-thin material, and is supported
only at its edges by a support frame or other suitable structure
16. The support frame can itself be a self-supported membrane.
There is no limitation on the thickness or configuration of the
support frame. But the membrane material 12 in which one or more
nanopores are to be formed is not mechanically supported at
interior regions of the membrane, thereby providing a free-standing
region in analogy with a trampoline.
[0019] The membrane 12 can optionally include one or more sites 18,
20, at which a material condition, such as a physical defect, is
included to effect the nucleation of a nanopore at the site. Such
sites can be produced, e.g., by inducing a crystal lattice defect,
such as a crystal lattice vacancy, at a selected location in the
membrane. For example, ion beam bombardment, electron beam
bombardment, UV ozone etch, patterned etching or chemical
treatment, or other process can be employed to form one or more
sites of a defect or other material condition that encourages
nucleation of a nanopore at that site.
[0020] As shown in FIG. 1, the membrane of atomically-thin material
is arranged in a liquid 14 that includes an electrically conducting
solution. In contact with the solution are provided electrically
conducting electrodes 22, 24, that are connected in a circuit 26
for applying an electrical potential, V.sub.in, between the
electrodes, across the membrane. The membrane is therefore
preferably configured between the electrodes 22, 24, so that the
electrodes are on opposite sides of the membrane 12.
[0021] Microelectronic substrates, wafers, and other support
structures can be employed for holding the membrane material in a
suitable configuration for nanopore formation. It can be
particularly efficient to produce the membrane in a configuration
in which the membrane will be operated for an intended application
after nanopore fabrication in the membrane. In one example of such,
conventional microelectronic materials are employed to arrange a
fabrication environment for a graphene membrane. The graphene
membrane material can be synthesized or provided in any suitable
manner. For example, single layer graphene can be grown by chemical
vapor deposition on a suitable substrate, such as a 25 .mu.m-thick
copper foil, at a temperature of, e.g., about 1000.degree. C. The
copper is first reduced under flow of hydrogen e.g., 20 sccm,
pumped with a scroll pump, for 30 minutes, then methane gas is
introduced at a flow rate of 40 sccm as a carbon source. Growth of
a graphene layer is conducted under these conditions in about 45
minutes. Exfoliated, epitaxially-grown, or other types of graphene
can also be used, and as explained above, other very thin membrane
materials, such as multilayer graphene, coated graphene, or boron
nitride, can also be used.
[0022] Referring to FIGS. 2A-2B, for many membrane applications, a
support frame structure 23 can provide the necessary mechanical
arrangement for both nanopore fabrication and subsequent operation.
In one example support frame structure 23, there is provided a
substrate, such as a conventional silicon wafer of 500 .mu.m in
thickness. A membrane support frame is provided on the substrate,
formed of, e.g., multiple material layers, such a 50 nm-thick
silicon nitride layer atop a 2 .mu.m-thick silicon oxide layer. As
shown in the cross-sectional view of FIG. 2A, each of the layers
includes an aperture, with the aperture being smallest in the
silicon nitride layer and largest in the silicon substrate. As a
result, as shown in FIG. 2B, there is provided a silicon nitride
membrane having a central aperture, shown as circular in FIG. 2B,
the edges of the silicon nitride membrane being supported by a
silicon nitride/silicon oxide membrane, the edges of which are
support by the silicon substrate. The atomically-thin membrane to
be processed is disposed on the surface of the silicon nitride. In
one example fabrication process for producing such, the layers of
silicon nitride and silicon oxide are blanket coated on the silicon
substrate, and with LTV lithography and reactive ion etching a
window, such as a 650 .mu.m-square window, one per 3 mm die, is
formed in the top silicon nitride layer on one side of the wafer. A
potassium hydroxide etch is used to etch the silicon down to the
oxide layer from the back side of the wafer, releasing a
free-standing silicon nitride/silicon dioxide membrane. Focused ion
beam milling is used to etch a .about.3 .mu.m window through
.about.90% of the oxide, followed by a hydrogen fluoride etch,
which releases a .about.3 .mu.m freestanding silicon nitride
membrane.
[0023] Using a suitable process, such as focused ion beam milling,
a circular aperture of between about 100 nm and about 200 nm in
diameter is then etched through the silicon nitride membrane. The
size of the hole can be varied from a few nanometers to a few
microns, above which an atomically-thin material such as graphene
will likely not be sufficiently mechanically supported. Other, more
scalable methods, such as lithography with reactive ion etching,
can be employed rather than sequential ion milling of multiple
apertures. The silicon nitride membranes with apertures are
preferably cleaned, e.g., with an RCA etch, i.e., ammonium
hydroxide, deionized water and 30% hydrogen peroxide in a 5:1:1
volume ratio at 70.degree. C. for 15 minutes, followed by a
ammonium fluoride dip, e.g., at room temperature for 1 minute, and
finally hydrochloric acid, deionized water, and 30% hydrogen
peroxide in a 5:1:1 volume ratio at 70.degree. C. for 15 minutes.
With this cleaning step, a selected atomically-thin membrane
material can be provided at the site of the support frame
structure.
[0024] For many atomically-thin materials, such as graphene, it can
be required to synthesize the material on a growth host and then
transfer the material to a support frame structure. In one example
membrane material transfer process, here for graphene, a graphene
layer that is synthesized on, e.g., copper foil is transferred to a
support frame by way of a support layer. Such a support layer can
be formed of any suitable material, e.g., methyl methacrylate
(MMA), 6-8% by weight in ethyl lactate, which can be spin-coated on
the to surface of a graphene layer as-synthesized on a copper foil,
and hard baked for about 5 minutes at 150.degree. C. The copper
foil can then be completely etched away, e.g., in an iron chloride
bath, by floating the foil on the surface, leaving a graphene
region with underlying MMA support layer. The graphene region is
then rinsed, with the floating graphene side facing the liquid,
preferably in a series of rinses, such as a two rinses in a 1 molar
hydrochloric acid bath and three rinses in deionized water. The
graphene is left floating on the air-water interface in the last
rinse. Then, a piece of the support frame structure wafer,
including e.g., 9 or more support frame die, is used to scoop up
the graphene region on top of the silicon nitride layer surface, so
as to cover the maximum area on the dies. Any water existing
between the graphene region and silicon nitride surface can be
expelled with a nitrogen gun. It is then preferred to employ a
bake, e.g., 95.degree. bake, of the entire structure, to ensure
that all water is removed and the graphene is well-bonded, by Van
der Waals forces, to the top surface of the silicon nitride layer.
Finally, the MMA support layer is removed by rinsing the structure
acetone, e.g., at a temperature of about 50.degree. C. for about 30
minutes. The entire structure is then rinsed in ethanol and dried
with a critical point dryer. Other methods for preparing a graphene
membrane can be employed, and for other atomically-thin materials,
other assembly protocols can be required. No particular membrane
material preparation is required; but it is preferred that the
membrane material be provided in a fixed arrangement on a support
frame structure.
[0025] With this support structure complete, a fluidic environment
including the atomically-thin membrane can be implemented in any
suitable manner. One example implementation of a suitable fluidic
cell 30 is shown schematically in FIG. 3. The fluidic cell includes
two half cells, each formed of a suitable inert material, such as
polyetheretherketone (PEEK), forming two microliter volume
reservoirs 32, 34. Each reservoir includes suitable fluidic inlets
36 and fluidic outlets 38, injection ports 40 for introducing
species into the reservoirs, and electrodes 22, 24 that can be
connected in a circuit for applying a voltage between the
reservoirs. For example, silver chloride electrodes can be screwed
into electrode ports via plastic fittings. Plastic gaskets 42, 44
of suitable material, e.g., molded polydimethylsiloxane (PDMS), are
disposed between the half cells, with a support structure including
the atomically-thin membrane 12 between the gaskets. The gaskets
42, 44 optionally can include one or more apertures 46 each having
a diameter, for example, of between about 10 .mu.m and about 100
.mu.m. The gasket apertures 46 are provided to reduce the area of
reservoir liquid in contact with the membrane 12 while providing
sufficient liquid contact, in an effort to reduce the device
capacitance. In assembly, the two half cells are brought together
to encase the membrane and its support structure between the
gaskets, fixing the two half cells in place relative to the
membrane.
[0026] The liquid in the reservoir of each half cell is preferably
highly electrically conducting. For many applications, an
electrolytic solution including a suitable concentration of
electrolyte, such as 1 M-3 M of a common electrolyte like KCl or
NaCl, is well-suited for the nanopore fabrication process. Any
suitable electrically conducting solution can be employed. The
solution can include species, such as molecules, like polymer
molecules for a selected nanopore application, such as DNA
sequencing or molecular analysis, so that upon nanopore
fabrication, the nanopore can be immediately operated in an
intended application.
[0027] For many membrane materials, it is found that the
characteristics of the membrane material surface can prohibit
complete wetting of the membrane surface when the membrane is
arranged in the electrically conducting solution. Herein is
provided a wetting technique for ensuring that both surfaces of the
membrane sufficiently wet in a solution such as an electrolytic
solution. In the wetting technique, a local environment in which
wetting of a surface is desired, e.g., a fluidic environment vessel
such as a flow cell arrangement like that of FIG. 3, is processed
with a series of wetting steps that enable substantially complete
wetting of the surface with the surface in place in the fluidic
environment.
[0028] In a first wetting step, the local wetting environment, such
as the flow cell, is flushed with a selected flushing gas, such as
high-purity carbon dioxide gas, at a sufficient pressure and for a
flushing duration that flushes substantially all of the air out of
the wetting environment, such as the flow cell. A gas flushing
duration of at least a few seconds, and up to about five minutes,
can be suitable for most configurations. After the cell is flushed
with the selected flushing gas, the flow cell and gas lines are
submerged in deionized, degassed water to prevent any air from
flowing back into the cell. Then in a second wetting step, while
submerged in the deionized water, a selected wetting solution is
introduced to the flow cell. The wetting solution is selected in
tandem with the selected flushing gas so that upon contact with the
flushing gas after introduction into the flow cell, the wetting
solution spontaneously reacts with the flushing gas to consume the
flushing gas or the flushing gas substantially completely dissolves
in the wetting solution.
[0029] For example, given a flushing gas of carbon dioxide, there
can be employed an aqueous wetting solution including potassium
hydroxide (KOH). The KOH in the wetting solution spontaneously
reacts with the CO.sub.2 flushing gas in a reaction as
2KOH+CO.sub.2.fwdarw.K.sub.2CO.sub.3+H.sub.2O. The K.sub.2CO.sub.3
reaction product including carbonate (CO.sub.3.sup.2-) ions is
highly soluble in water, and immediately dissolves in the KOH
solution. After this reaction is initiated, any residual carbon
dioxide gas bubbles remaining in the system, and particularly at
the surface of the membrane, are consumed by the solution-gas
reaction, and the membrane surface thereby completely wets. For
example, with a 10 mM KOH solution at 1 atmosphere of pressure and
room temperature, the rate of absorption of a CO.sub.2 gas bubble
is about 0.1 .mu.L/secmm.sup.2, indicating that any CO.sub.2
bubbles smaller than .about.1 .mu.l that remain at a membrane
surface will dissolve by reaction with KOH in solution within 1
min, thereby quickly achieving complete wetting of the membrane
surface.
[0030] In this membrane surface wetting procedure, the wetting
solution is flowed into the flow cell against the flushing gas
pressure, and the flow of flushing gas can then be turned off at a
suitable time, such as once the gas-solution reaction is initiated.
After a suitable gas-solution reaction duration, e.g., a few
minutes, the wetting process is complete and the wetting solution
can be replaced with any selected solution for which surface
wetting is desired. No air or flushing gas bubbles are introduced,
and the membrane surface remains completely wetted. No processing
of the membrane surface is required to maintain the wetted
condition.
[0031] This wetting method is inexpensive, very fast and efficient,
and safe, and enables complete wetting of fragile hydrophobic
materials, such as graphene membranes, that can be physically
destroyed by conventional hydrophilic treatments. This wetting
procedure enables the wetting of graphene surfaces and graphene
nanopores with a yield of >95%. The wetting method is applicable
to any micro-scale or nano-scale system in which surface wetting is
required.
[0032] This surface wetting method is not limited to the CO.sub.2
flushing gas and KOH aqueous solutions described above. Any
flushing gas-wetting solution pair can be employed in which the
flushing gas either reacts with the wetting solution to form a
soluble reaction product or itself dissolves in the wetting
solution. For example, the CO.sub.2 flushing gas can be employed
with any wetting solution that is pH basic, such as NaOH or
NH.sub.4OH. Similarly, Cl.sub.2 flushing gas can be employed with a
basic wetting solution such as KOH, NaOH, or NH.sub.4OH, whereby
reaction product ClO.sup.- ions can be dissolved in solution upon
reaction between the Cl.sub.2 and the solution. Any suitable
flushing gas can be employed, including, e.g., ammonia gas or other
selected gas, along with a selected companion wetting solution that
operates to remove flushing gas from the
[0033] Other surface wetting techniques can be employed; no
particular wetting process is required. It is, however, preferable
that both sides of the membrane surface be fully wetted and in full
contact with the electrically conducting solution. Whatever wetting
process is employed, it is preferable to confirm the compatibility
of the process gases and liquids with a membrane material under
consideration.
[0034] Once the membrane is wetted in the liquid flow cell solution
with a selected electrically conducting liquid, there is initiated
the procedure of nucleation of a nanopore or nanopores in the
membrane. In a first step nucleation, the electrical leakage
conductance of the membrane is measured. The leakage conductance
can be measured with any suitable technique. In one example
technique, a voltage ramp of suitable amplitude, such as -150 mV to
+150 mV over 10 seconds, can be imposed across the membrane at the
flow cell electrodes, e.g., using a signal generator, and the
resulting current through the system, between the electrodes across
the membrane, is measured, e.g., with a current amplifier connected
in series with the membrane. It is preferred that the voltage
amplitude applied in measuring leakage conductance be relatively
low, e.g., no greater than about .+-.300 mV, to ensure that the
measurement does not disrupt the membrane surface.
[0035] Referring to FIG. 4A, once the leakage conductance is
determined, then one or more nanopores are nucleated in the
membrane by applying at least one ultra-short nucleation voltage
pulse, and for many applications, preferably a sequence of
ultra-short nucleation voltage pulses, between the electrodes in
the solution. Each nucleation voltage pulse has a pulse duration,
d, that can be the same duration as that of other nucleation pulses
in the pulse sequence, or that can be a distinct pulse duration.
The duration of each nucleation voltage pulse is no greater than
about 0.00001 seconds, or 10.times.10.sup.-6 seconds, and for many
applications, is preferably no greater than about
500.times.10.sup.-9 seconds and for some applications is preferably
no greater than 250.times.10.sup.-9 seconds. The duration of each
nucleation voltage pulse is at least about 100.times.10.sup.-9
seconds to accommodate RC charging in the system across the
membrane. With these requirements, each nucleation voltage pulse
duration is at least about 100,000 times shorter than one
second.
[0036] The amplitude of the first nucleation voltage pulse in a
sequence of ultra-short voltage pulses is set at a starting
voltage, V.sub.S, and thereafter, the voltage of each subsequent
nucleation voltage pulse can be increased or can repeat a previous
voltage level. The first voltage pulse is set at a voltage below a
minimum voltage that is expected for nucleating a nanopore, e.g., 1
V, and the voltage amplitude of each subsequent pulse can be
incrementally increased by a relatively small value, e.g., 1 V or
less, such as 100 mV. Between each nucleation voltage pulse
application, there is a pause in nucleation voltage pulse
application of at least about the duration of the RC time constant
for the system, e.g., 10.times.10.sup.-9 seconds.
[0037] After a first nucleation voltage pulse and any subsequent
pulses that are included, it can be preferred to measure the
conductance of the membrane to determine if a nanopore has
nucleated in the membrane. If after a nucleation voltage pulse the
conductance of the membrane is found to have increased over the
leakage conductance measured earlier, then the nucleation of a
nanopore is indicated; for example, if the measured conductance is
found to have increased appreciably, e.g., to at least about, e.g.
1 nS to 10 nS, here for an example of a liquid 1M KCl solution,
then nanopore nucleation is confirmed. The site of a nucleated
nanopore is physically characterized by an extent that can be less
than 1 nm. For many materials, this nucleated nanopore extends
through the thickness of the membrane, but may not extend through
the membrane thickness for all materials. Once nanopore nucleation
is confirmed, no additional nanopore nucleation voltage pulses are
required. This nanopore nucleation confirmation can occur after one
nanopore nucleation voltage pulse is applied or after two or more
nanopore nucleation voltage pulses are applied.
[0038] In making a membrane conductance measurement to confirm
nanopore nucleation, it can be preferred to employ a relatively
low-amplitude pulse of voltage, e.g., a voltage less than about
.+-.300 mV, to ensure that the conductance measurement does not
alter any nanopore that may have nucleated during a previous
voltage pulse. At such low voltages, no damage is done to the
membrane or nanopore, so measurements 1 sec or longer can be used
to accurately measure conductance.
[0039] Once it is confirmed that one or more nanopores have
nucleated in the membrane, then if desired, the diameter of the
nanopore can be enlarged by removal of material from the membrane
at edges of the nucleated nanopore. Such nanopore diameter
enlargement is not required, and may not be necessary for a given
application, but for many applications, can be employed for tuning
the nucleated nanopore diameter. Any suitable process can be
employed to enlarge a nanopore diameter. For example, membrane
etching, by liquid, vapor, plasma, or other species, can be
employed to remove membrane material from nanopore edges. The
nanopore diameter can be tuned to a prespecified selected nanopore
diameter that is desired for the nucleated nanopore. No particular
nanopore diameter tuning method is required and indeed, as just
explained, for some applications nanopore nucleation can be
sufficient.
[0040] In one elegantly convenient technique nanopore diameter
tuning technique, referring back to FIG. 1, a sequence of
ultra-short voltage pulses is applied between the electrodes in
solution in the manner described above for nucleating a nanopore.
With this technique, the nanopore diameter can be tuned to an a
priori prespecified nanopore diameter, as described in detail
below, or can be enlarged in an open loop fashion. In either
scenario, there is applied at least one ultra-short tuning voltage
pulse, or for many applications, a sequence of ultra-short tuning
voltage pulses.
[0041] Referring to FIG. 4B, in such a sequence of tuning voltage
pulses, the first tuning voltage pulse has a pulse amplitude,
V.sub.s, that is similar to the starting amplitude of the first
nucleation voltage pulse for nucleating a nanopore. Each subsequent
tuning voltage pulse amplitude can be greater than that of previous
pulses or can provide consecutive pulses of equal voltage
amplitude. Each tuning voltage pulse has a pulse duration, d, that
can be the same duration as that of other tuning voltage pulses in
the pulse sequence, or that can be a distinct pulse duration. The
duration of each tuning voltage pulse can be set to any suitable
value. For many applications, it can be preferred that a tuning
voltage pulse duration that is no greater than about 0.00001
seconds, or 10.times.10.sup.-6 seconds, and for many applications,
is preferably no greater than about 500.times.10.sup.-9 seconds.
The duration of each tuning voltage pulse is at least about
100.times.10.sup.-9 seconds to accommodate RC charging in the
system across the membrane. With these requirements, each tuning
voltage pulse duration is at least about 100,000 times shorter than
one second. Each tuning pulse duration can be the same, or can be
shorter than prior pulses as a desired nanopore diameter is neared.
Each nanopore nucleation pulse and each nanopore tuning pulse can
be of the same duration.
[0042] It is discovered in the nanopore fabrication process herein
that the duration of the voltage pulses for both nanopore diameter
enlargement as well as for nanopore nucleation are preferably
shorter than about 0.00001 seconds, or 10.times.10.sup.-6 seconds,
and for many applications, the pulse duration is preferably shorter
than about 500.times.10.sup.-9 seconds or 250.times.10.sup.-9
seconds. Any voltage pulse duration longer than about 0.00001
seconds can damage the atomically-thin membrane, and a voltage
application of as long as one second can be expected to locally
destroy the atomically-thin membrane. A continuous voltage
application, even when ramped in voltage amplitude, cannot be
applied. Instead, it is discovered that to preserve the mechanical
integrity of the atomically-thin membrane, voltage pulses having a
duration that is at least 100,000 times shorter than one second are
required.
[0043] It is discovered herein that nanopores smaller than 1 nm in
diameter can be created and controllably enlarged in atomically
thin membranes with subnanometer precision by applying an electric
field that is not localized on the nanometer scale. Indeed,
continuous voltage application destroys the mechanical integrity of
atomically-thin membranes so quickly that feedback measurement of
the pore current cannot be used to control pore size on the
nanometer scale. Only the application of voltage pulses having a
pulse duration within a very small pulse duration range that is
between the RC charging time of the membrane, which is about
10.sup.-7 seconds, and the time scale of membrane destruction,
which can be 10.sup.-6 seconds or less, allows one to arrest this
violent process in a way that affords precise control of pore size
on the nanoscale.
[0044] The nanopore diameter can be precisely tuned to a
prespecified diameter by calibrating nanopore sizes with
corresponding membrane electrical conductance values for a
specified membrane material and electrolytic solution. Here the
conductance of a given membrane in a selected electrolytic solution
can be calculated as a function of nanopore size in the membrane
by, e.g., finite element simulation and analysis, or by analytical
approximation. Alternatively, nanopores can be physically formed
by, e.g., electron beam drilling, in a selected membrane material
and the conductance of the nanopores experimentally measured as a
function of nanopore size in a selected electrolyte solution. For
many atomically-thin membranes, such as single layer graphene, the
conductance of the membrane scales linearly with nanopore diameter.
For example, in a 1 M KCl solution, the nanopore diameter in
Angstroms is approximately equal to the conductance in
nanoSiemens.
[0045] With a known correlation between membrane conductance and
nanopore diameter, the nanopore tuning method can be precisely
controlled for a calibrated membrane material and electrolytic
solution. After each tuning voltage pulse applied to a nucleated
nanopore, a measurement voltage pulse can be applied to measure the
conductance of the membrane for assessing the extent of the
nanopore. The measurement voltage pulse is preferably of a
relatively low voltage amplitude, e.g., about 300 mV, applied in
the manner described above, so as to not affect the state of the
nanopore. With such a measurement pulse, the membrane conductance
can be measured for determination of nanopore diameter.
[0046] After a membrane conductance measurement, a subsequent
nanopore diameter tuning voltage pulse can be applied to the
electrodes, with the subsequent tuning pulse providing increased
voltage amplitude or constant voltage amplitude. The voltage pulse
amplitude can be increased from one tuning voltage pulse to the
next until an increase in membrane conductance is observed,
indicating that the nucleated pore diameter has increased. Once it
is determined that the nucleated pore extent is increasing, the
tuning voltage pulse amplitude can be held constant for subsequent
tuning voltage pulses, in the manner shown in FIG. 3B for 5
V-amplitude voltage pulses. As the nanopore enlarges, it can be
preferred to vary the pulse duration, d, to increase the precision
of control over nanopore growth. The tuning pulse duration, d, can
therefore be decreased over the pulse sequence as the nanopore
diameter increases.
[0047] It is discovered in the nanopore fabrication process herein
that if a membrane conductance measurement is employed to determine
the conditions of nanopore nucleation and/or enlargement, such a
membrane conductance measurement must be made between applied
voltage pulses. It is not feasible to monitor the system
conductance while a voltage pulse is being applied with the intent
of ending the voltage pulse when a desired nanopore diameter is
attained. The time scale in which a nanopore enlarges in the
nanometer range is on the order of the RC charging time of the
membrane. During the duration of the RC charging time, the current
through a nanopore cannot be measured accurately because it is
dominated by the capacitive current originating from the voltage
pulse. As a result, the current through the nanopore, and
consequently, the nanopore size, cannot be measured before it has
grown far beyond the desired size. It is therefore preferred that
each ultra-short voltage pulse be applied without monitoring the
system conductance during the voltage pulse application, after
which the nanopore conductance can measured with a low voltage
bias, and can be safely applied for relatively long times without
destroying the membrane.
[0048] The nanopore tuning process can be fully calibrated for a
selected membrane material and other system parameters and then
conducted in an open-loop fashion without conductance measurement
during the process. For example, one or more nanopore nucleation
voltage pulses can be applied with preselected pulse amplitude and
duration that are known to produce nanopore nucleation, and then a
selected process can be undertaken to tune the diameter of the
nucleated nanopore. For example, one or more nanopore tuning
voltage pulses can be applied with preselected pulse amplitude and
duration that are known to produce a prespecified nanopore
diameter. This process can include, e.g., one nanopore nucleation
voltage pulse and one nanopore tuning voltage pulse, with each
pulse prespecified for ultimately producing a selected nanopore
diameter.
[0049] As explained above, any suitable process can be employed to
enlarge a nanopore diameter once nanopore nucleation is confirmed.
Application of voltage pulses in the manner described above can be
particularly efficient and effective, but other processes can be
employed. For example, chemical species can be introduced to the
liquid environment to chemically enlarge a nucleated nanopore. In
one example of such, potassium permanganate in sulfuric acid
solution is introduced into a liquid environment in which a
graphene membrane and nanopore are disposed to react with the
graphene nanopore walls and enlarge the nanopore in the graphene.
In other examples, the temperature of the liquid environment can be
controlled to tune a nanopore size. For example, the liquid can be
heated to enlarge a nanopore. In addition, there can be employed a
range of electrical processes that tune a nanopore diameter, e.g.,
to enlarge a nanopore.
[0050] Beyond nanopore tuning, after nanopore nucleation and/or
diameter tuning the membrane can be further processed for any
particular requirements. For example, the membrane and/or nanopore
can be coated with materials by chemical or physical deposition,
such as atomic layer deposition. Such material coating can include
microelectronic materials, such as electrically insulating
materials, and can include molecules, such as lipid molecules,
employed for a selected application. These examples demonstrate
that once a nanopore is nucleated in a membrane by one or more
ultra-short voltage pulses, a wide range of processes can be
employed to tune the nanopore diameter and to customize the local
materials and structure at the site of the nanopore.
[0051] With these fabrication processes and surface wetting
technique, a nanopore is precisely formed in-solution and remains
wetted in solution at the conclusion of the nanopore nucleation and
tuning steps, and thus can immediately be used for solution
measurements without a need for removal from the solution
environment. The surface of the membrane can be maintained in
pristine condition, without contamination that could occur by
ambient exposure out of the solution. Thereby are enabled highly
precise nanopore applications, such as DNA translocation through
the nanopore.
Example I
Nanopore Nucleation and Enlargement in a Graphene Membrane
[0052] A graphene membrane was assembled with a support frame
structure of silicon nitride, silicon oxide, and a silicon
substrate, as shown in FIGS. 2A-2B. The support structure was
arranged in a flow cell like that of FIG. 3, configured with 3 M
KCl solution at room temperature. A nanopore nucleation and tuning
process was conducted in the manner described above. FIG. 5 is a
plot of the voltage amplitude of each applied voltage and the
current that was measured between each voltage pulse. Each voltage
pulse, both in the nucleation voltage sequence and the tuning
voltage sequence, was 250.times.10.sup.-9 seconds in duration.
[0053] A low voltage bias of 160 mV was applied across the membrane
for conductance measurement, and conductance was measured using an
Axopatch 200B low noise current amplifier. An Arduino Uno R3 board
was programmed to allow manual control of the nanopore fabrication
process. In response to a button manually pressed on the Arduino
Uno, the membrane was disconnected from the Axopatch current
amplifier and connected to an HP 8110A pulse generator by way of a
mechanical relay (Panasonic TN2-5V). After a delay of 1 second, the
Arduino board triggered the pulse generator, which applied a pulse
of 250 ns with manually-specified voltage amplitude, and
reconnected the membrane to the low noise current amplifier to
monitor the nanopore size. These steps were repeated several times
as indicated in the diagram.
[0054] As shown in the plot of FIG. 5, after an application of
seven nucleation voltage pulses, starting at 1 V in amplitude and
increasing to 7 V in amplitude, there was nucleated a nanopore, as
evidenced by a jump in measured current. A sequence of tuning
voltage pulses was then initiated, beginning again with a starting
voltage of 1 V. After application of five tuning voltage pulses,
increasing in amplitude from 1 V to 5 V, the nanopore began to
enlarge, as evidenced by a jump in measured current. After the
measured jump in current, the tuning voltage pulse amplitude was
held constant at 5 V and the sequence of tuning voltage pulses was
continued. As shown in the plot, the measured conductance continued
to increase as the tuning voltage pulse sequence continued,
indicating an increase in nanopore diameter. FIG. 6 is a plot of
calculated nanopore diameter as a function of voltage pulse number,
including nucleation voltage pulses and tuning voltage pulses.
[0055] In making observations of a nanopore in an atomically-thin
material, it is found that imaging of a nanopore can be difficult
because the image contrast can be poor, particularly for
single-layer graphene. A transmission electron microscope (TEM) is
necessary, and aberration corrected microscope at low energies (80
kV) is very helpful. In one process for successfully imaging a
nanopore in a graphene membrane, the graphene membrane as-arranged
on a support structure is first carefully removed from a flow cell
and quickly immersed in deionized water. The membrane and support
structure should not be allowed to dry in air. At a time for
imaging the nanopore, the membrane and support structure should be
dried with a critical point dryer. Contaminants can cover a
nanopore during the drying process and exposure to ambient air,
making imaging unsuccessful. In addition, imaging with a beam
having an energy above about 200 kV can gradually induce defects in
an atomically-thin material such as single-layer graphene.
Therefore, it is preferred to very quickly obtain an image. Clean
graphene often looks similar to vacuum, so interpretation of the
images is also difficult.
[0056] The conductance measurement data of FIG. 5 demonstrate that
with nucleation and tuning voltage pulses of no more than
250.times.10.sup.-9 seconds in duration, a nanopore is nucleated in
a graphene membrane and the nanopore diameter is controllably and
precisely enlarged, pulse-by-pulse, to a desired diameter, in this
experimental example, 3 nm. The nanopore nucleation is distinctly
indicated by measured conductance values after each nucleation
voltage pulse, and nanopore diameter increase is clearly correlated
to measured conductance values after each tuning voltage pulse. The
nanopore fabrication method is demonstrated to reliably produce
nanopores while maintaining the mechanical integrity of the
membrane material.
Example II
DNA Translocation Through a Nanopore Fabricated in a Graphene
Membrane
[0057] The nanopore of Example I having a diameter of 3.5 nm was
positioned in the flow cell of FIG. 3 in a 3 M KCl solution.-10 kbp
dsDNA was dissolved in the electrolyte solution of 3 M KCl at a
concentration of 4 ng/.mu.L. 45 .mu.L of the solution including DNA
was injected into the injection port of the fluidic cell into the
half-cell on the top side of the structure, that being the side
having the graphene layer fully exposed on top of the silicon
nitride/silicon oxide support structure, as opposed to the
underside, on which the surface of the lower silicon substrate was
exposed. Using a patch-clamp amplifier (Axopatch 200B), a -160 mV
bias was applied to the electrode on the graphene layer side of the
structure. This voltage bias electrophoretically drove the DNA
molecules in the solution to translocate through the nanopore from
the graphene layer side of the structure to the silicon substrate
side of the structure. During the voltage application, the ionic
current through the nanopore was monitored using the patch-clamp
amplifier. DNA translocations were detected as transient current
blockades in the measured ionic current.
[0058] As shown in the plot of FIG. 7, each measured current
blockade consisted of a drop in measured current, for example, a
current drop of about 3.5 nA, over a duration of about
200.times.10.sup.-6 seconds. These conditions directly correspond
to a blockage of ionic current due to the presence of the DNA
molecule threaded through the nanopore. This DNA translocation
through the nanopore was conducted over several minutes. This
demonstrates that nanopores fabricated by ultra-short voltage pulse
in the methodology herein are sufficiently robust and well-defined
to enable DNA translocation through the nanopore over an extended
duration of operation.
[0059] The examples and description above demonstrate the precision
and high level of dimensional control that is achieved with the
nanopore fabrication process provided herein, providing
repeatability and elegant simplicity in its implementation. While
the process description has presented a scenario in the fabrication
of a single nanopore, the process is easily configured for
scalability. As explained above, a membrane material can be
provided with an array of surface sites each having a condition, or
defect, that encourages nanopore nucleation at the site, whereby an
array of nanopores can be fabricated simultaneously. Alternatively,
there can be employed arrays of electrodes, or electrodes that can
scan across the surface of the membrane material, as electrical
pulses are applied to the electrodes, to fabricate a plurality of
nanopores in the material, e.g., as a prespecified array of
nanopores. Such arrays of nanopores can be particularly
advantageous for, e.g., nanofiltration applications.
[0060] The nanopore fabrication process can be employed with any
suitable atomically-thin material or layers of material that
together are less than about ten atomic layers thick, or less than
about 50 nanometers-thick, less than about 10 nanometers thick, or
even less than about 5 nanometers-thick. The nanopore fabrication
process can be conducted in the presence of any selected species in
the liquid environment of the nanopore formation, including
chemically reactive species and species such as polymer molecules
that are to be examined after nanopore formation. Further, a
selected one or more chemical species can be included in the liquid
environment, during nanopore formation, for example, to modify the
edges of the nanopore during nanopore formation or to enlarge the
nanopore, as well as to modify or react with the membrane material.
Thus, the nanopore fabrication process is amenable to extensive
adaption for customizing a nanopore and its environment for a
selected application.
[0061] It is recognized that those skilled in the art may make
various modifications and additions to the embodiments described
above without departing from the spirit and scope of the present
contribution to the art. Accordingly, it is to be understood that
the protection sought to be afforded hereby should be deemed to
extend to the subject matter claims and all equivalents thereof
fairly within the scope of the invention.
* * * * *